Dye transfer inhibiting compositions

ABSTRACT

Dye transfer inhibiting compositions are disclosed, comprising an enzyme exhibiting peroxidase activity, a hydrogen peroxide or a hydrogen peroxide precursor or an enzymatic system capable of generating hydrogen peroxide, an additional oxidizable substrate, and a cellulase, characterized in that the cellulase provides at least 10% removal of immobilized radioactive labelled carboxymethyl cellulose according to C14CMC method at 25×10 -6  % by weight of the laundry test solution. 
     According to the present invention, a preferred cellulase consists essentially of a homogeneous endoglucanase component which is immunoreactive with a monoclonal antibody raised against a partially purified=43 kD cellulase derived from Humicola insolens DM1800.

FIELD OF THE INVENTION

The present invention relates to an enzymatic composition for inhibitingthe transfer of dye from a dyed fabric to another fabric during washing.

BACKGROUND OF THE INVENTION

One of the most persistent and troublesome problems arising duringmodern fabric laundering operations is the tendency of some coloredfabrics to release dye into the laundering solutions. The dye is thentransferred onto other fabrics being washed therewith.

One way of overcoming this problem would be to bleach the fugitive dyeswashed out of dyed fabrics before they have the opportunity to becomeattached to other articles in the wash.

Suspended or solubilized dyes can to some degree be oxidized in solutionby employing known bleaching agents.

GB 2 101 167 describes a stable liquid bleaching composition containinga hydrogen peroxide precursor which is activated to yield hydrogenperoxide on dilution.

However, it is important at the same time not to bleach the dyesactually remaining on the fabrics, that is, not to cause color damage.

U.S. Pat. No. 4,077,768 describes a process for inhibiting dye transferby the use of an oxidizing bleaching agent together with catalyticcompounds such as iron porphins.

U.S. patent application Ser. No. 07/421,414 now abandoned describesperoxidases and oxidases utilized for the oxidation of organic orinorganic substances, including coloured substances. A dye transferinhibiting composition comprising an enzymatic system capable ofgenerating hydrogen peroxide and iron catalysts has been disclosed incopending EP Patent Application 91202655.6 filed Oct. 9, 1991.

EP 424 398-A describes a detergent additive capable of exerting ableaching effect comprising a peroxidase. The additive further comprisesone or more enzymes, particularly a lipase, protease, amylase or acellulase.

EP-A-350 098 discloses a C14CMC-method which defines a cellulaseselection criteria relevant for detergent application. A minimum of 10%removal of immobilized radioactive labelled carboxymethylcelluloseaccording to the CMC-method at 25×10⁻⁶ % by weight of the cellulaseprotein in the test solution has been found to provide high activecellulase.

A preferred group of cellulase falling under the high activitydefinition according to the present invention has been disclosed incopending Danish Patent Application No. 1159/90 filed May 5, 1990. Thereis disclosed a cellulase preparation consisting essentially of ahomogeneous endoglucanase component which is immunoreactive with amonoclonal antibody raised against a partially purified=43 kD cellulasederived from Humicola insolens DM1800.

It has now been surprisingly found that the efficiency of peroxidases interms of dye transfer inhibition, is considerably enhanced by using saidhigh activity cellulase and more in particular the specific cellulasepreparation disclosed in copending Danish Patent Application No.1159/90. It is therefore an object of the present invention to providedye transfer inhibiting compositions which exhibit optimum dye transferin wash liquids by using said high activity cellulase and peroxidase.

According to one embodiment of this invention a dye transfer inhibitingcomposition is provided which comprises a cost-effective cellulasepreparation, e.g. by employing recombinant DNA techniques.

According to another embodiment of this invention a process is alsoprovided for laundering operations involving colored fabrics.

SUMMARY OF THE INVENTION

The present invention provides a dye transfer inhibiting compositioncomprising an enzyme exhibiting peroxidase activity, a hydrogen peroxideor a hydrogen peroxide precursor or an enzymatic system capable ofgenerating hydrogen peroxide, an additional oxidizable substrate, and acellulase, characterized in that the cellulase provides at least 10%removal of immobilized radioactive labelled carboxymethyl celluloseaccording to C14CMC method at 25×10⁻⁶ % by weight of the laundry testsolution.

According to the present invention, a preferred cellulase consistsessentially of a homogeneous endoglucanase component which isimmunoreactive with a monoclonal antibody raised against a partiallypurified=43 kD cellulase derived from Humicola insolens DM1800.

DETAILED DESCRIPTION OF THE INVENTION Cellulase

The activity of enzymes and particularly the activity of cellulaseenzyme has been defined for various applications by different analyticalmethods. These methods all attempt to provide a realistic assessment ofthe expected in use performance or at least a measurement correlatingwith the in use performance. As has been detailed in European PatentApplication EP-A-350098, many of the methods, particularly thesefrequently used by cellulase manufacturers, are not sufficientlycorrelated with the in use performance of cellulase in laundry detergentcompositions. This is due to the various other usage conditions forwhich these activity measurement methods have been developed.

The method described in EP-A-350098, has been developed to be and tohave a predictive correlation for the ranking of cellulase activity inlaundry detergent compositions.

The present invention therefore uses the method disclosed in EP-A-350098to screen cellulases in order to distinguish cellulases which are usefulin the present invention and those which would not provide theobjectives of the present invention. The screening method, hereinafterreferred to as C14CMC-Method, which has been adopted from the methoddisclosed in EP-A-350098, can be described as follows:

Principle

The principle of the C14CMC-Method for screening is to measure at adefined cellulase concentration in a wash solution the removal ofimmobilized carboxy methyl cellulose (CMC) from a cloth substrate. Theremoval of CMC is measured by radio-active labelling of some of the CMCby using C14 radio-active carbon. Simple counting of the amount ofradio-active C14 on the cloth substrate before and after the cellulasetreatment allows the evaluation of the cellulase activity.

Sample Preparation

CMC preparation: The radio-active CMC stock solution is preparedaccording to Table I. The radio-active CMC can be obtained by methodsreferred to in EP-A-350098.

Fabric substrates: The fabric substrates are muslin cotton swatcheshaving a size of 5 cm×5 cm. They are inocculated with 0.35 ml of theradio-active labelled CMC stock solution in their center. The muslincotton swatches are then airdried.

Immobilization of CMC: To immobilize the radio-active labelled CMC onthe muslin cotton swatches, laundero-meter equipment "Linitest OriginalHaunau" made by Original Haunau, Germany, is used. A metal jar of thelaundero-meter is filled with 400 ml of hard water (4 mmol/liter of Ca⁺⁺ions). A maximum number of 13 swatches can be used per jar. The jar isthen incubated in a heat-up cycle from 20° C. to 60° C. over 40 minutesin the laundero-meter equipment. After incubation the swatches arerinsed under running city water for 1 minute. They are squeezed andallowed to airdry for at least 30 minutes. According to EP-A-350098samples of the swatches with immobilized radio-active CMC can also bemeasured as "blank samples" without washing.

Sample Treatment

Laundry test solution: The laundry test solution is prepared accordingto the composition of Table II. It is balanced to pH 7.5. The laundrytest solution is the basis to which a cellulase test sample is added.Care should be taken to not dilute the laundry test solution by addingwater to a 100% balance prior to having determined the amount ofcellulase to be added. The amount of cellulase which is used in thisscreening test should be added to provide 25×10⁻⁶ weight percent ofcellulase protein in the laundry test solution (equivalent to 0.25milligram/liter at 14.5° C.).

Wash procedure: The swatches thus inocculated with radio-active labelledCMC are then treated in a laundry simulation process. The laundryprocess is simulated in the laundero-meter type equipment, "Linitest,Original Haunau", by Original Haunau, Haunau Germany An individualswatch is put into a 20 cm³ glass vial. The vial is filled with 10 ml ofthe laundry test solution and then sealed liquid tight. Up to 5 vialsare put into each laundero-meter jar. The jar is filled with water as aheat tranfer medium for the laundering simulation. The launderingsimulation is conducted as a heat-up cycle from 20° C. to 60° C. over 40minutes.

After the processing of the samples the vials are submerged in coldwater and subsequently each swatch is taken out of its vial, rinsed in abeaker under running soft water, squeezed and allowed to airdry for atleast 30 minutes.

Measurement

In order to measure radio-active labelled CMC removal, a scintillationcounter, for example, a LKB 1210 Ultrabeta Scintillation Counter, isused. In order to obtain most accurate results, the instruction manualfor optimum operation of the particular scintillation counter should befollowed. For example, for the LKB 1210 Ultrabeta Scintillation Counter,the following procedure should be followed. The swatch to be measured isput into a plastic vial filled with 12 ml of scintillator liquid (e.g.scintillator 299 from Packard). The swatch is then allowed to stabilizefor at least 30 minutes. The vial is then put into the LKB 1210Ultrabeta Scintillation Counter and the respective radio-activity countsfor the swatch is obtained.

In order to measure the amount of CMC removal due only to the cellulase,a measurement of a swatch which has been inocculated at the same timebut has been treated in the laundry test solution without cellulase, isnecessary. The activity of the cellulase is then expressed as percent ofradio-active labelled CMC removal. This percentage is calculated by thefollowing formula: ##EQU1## Wherein XO is the radioactivityscintillation count of a swatch treated with the laundry test solutionwithout cellulase

XC is the radioactivity scintillation count of a swatch treated with thelaundry test solution containing the cellulase to be evaluated

Statistical Considerations, Procedure Confirmation

In order to provide statistically sound results, standard statisticalanalysis should be employed. For the given example, using the LKB 1210Ultrabeta Scintillation Counter, it has been found that a sample size of3 swatches for each radioactivity scintillation count can be used.

In order to confirm the procedure by internal crosschecking, measurementand calculation of the "blank sample" according to EP-A-350098 arerecommended. This will allow to detect and eliminate errors.

Interpretation of Results

The described screening test does provide a fast, unique and reliablemethod to identify cellulases which satisfy the activity criteria of thepresent invention versus cellulases which are not part of the presentinvention.

It has been found that a removal of 10% or more of the immobilizedradioactive labelled CMC according to the above C14CMC-method, indicatesthat the respective cellulase satisfies the requirements of theinvention.

It will be obvious to those skilled in the art that removal percentagesabove 10% indicate a higher activity for the respective cellulase. Ittherefore is contemplated that cellulase providing above 25% orpreferably above 50% removal of radioactive labelled CMC, at the proteinconcentration in the laundry test solution according to theC14CMC-method, would provide indication of an even better performance ofthe cellulase for use in laundry detergents.

It also has been contemplated that usage of higher concentrations ofcellulase for C14CMC-method, would provide higher removal percentages.However, there exists no linear proven correlation between cellulaseconcentration and removal percentage obtained by it.

It also has been contemplated that usage of higher concentrations ofcellulase for C14CMC-method, would provide higher removal percentages.

                  TABLE I                                                         ______________________________________                                        Radioactive C.sub.14 labelled CMC stock solution                              (all percentages by weight of total solution)                                 ______________________________________                                        Total CMC*         99.2 × 10.sup.-3 %                                   (CMC should be                                                                detergent grade CMC                                                           with a degree of                                                              substitution from about                                                       0.47 to about 0.7)                                                            Ethanol          14985.12 × 10.sup.-3 %                                 Deionized Water  84915.68 × 10.sup.-3 %                                 Total:           100%                                                         ______________________________________                                         *Total CMC contains nonradioactive and radioactive CMC to provide a           radioactivity which allows sufficiently clear readings on the                 scintillation counter used. For example, the radioactive CMC can have an      activity of 0.7 millicurie/g and be mixed with nonradioactive CMC at a        ratio of 1:6.7.                                                          

                  TABLE II                                                        ______________________________________                                        Laundry test solution                                                         (all percentages by weight of total solution)                                 ______________________________________                                        Linear C.sub.12 alkyl                                                                          0.110%                                                       benzene sulphonic acid                                                        Coconut alkyl sulphate                                                                         0.040%                                                       (TEA salt)                                                                    C.sub.12-15 alcohol                                                                            0.100%                                                       ethoxylate (E07)                                                              Coconut fatty acid                                                                             0.100%                                                       Oleic acid       0.050%                                                       Citric acid      0.010%                                                       Triethanolamine  0.040%                                                       Ethanol          0.060%                                                       Propanediol      0.015%                                                       Sodium hydroxide 0.030%                                                       Sodium formate   0.010%                                                       Protease         0.006%                                                       Water (2.5 mmol/liter                                                                          balance to 100%                                              Ca.sup.++), pH adjustment                                                     agent (HCL or NaOH                                                            solutions) and                                                                cellulose                                                                     ______________________________________                                    

According to the present invention, preferred cellulases are those asdescribed in Danish Patent Application 1159/90. For example, a cellulasepreparation useful in the compositions of the invention can consistessentially of a homogeneous endoglucanase component, which isimmunoreactive with an antibody raised against a highly purified 43 kDcellulase derived from Humicola insolens, DSM 1800, or which ishomologous to said 43 kD endoglucanase.

It should be stressed that all cellulase enzymes according to thepresent invention have to meet the criteria of the above mentionedscreening test. However, in the Danish Patent Application 1159/90additional criteria are established allowing to identify preferredcellulase enzymes in combination with present screening test.

Cellulase preparations particulary useful in the compositions of theinvention are those in which in addition to the screening test, theendoglucanase component exhibits a CMC-endoase activity of at leastabout 50, preferably at least about 60, in particular at least about 90CMC-endoase units per mg of total protein. In particular, a preferredendoglucanase component exhibits a CMC-endoase activity of at least 100CMC-endoase units per mg of total protein.

In the present context, the term "CMC-endoase activity" (cevu) refers tothe endoglucanase activity of the endoglucanase component in terms ofits ability to degrade cellulose to glucose, cellobiose and triose, asdetermined by a viscosity decrease of a solution of carboxymethylcellulose (CMC) after incubation with the cellulase preparation of theinvention, as described in detail below.

The CMC-endoase (endoglucanase) activity can be determined from theviscosity decrease of CMC, as follows: A substrate solution is prepared,containing 35 g/l CMC (Hercules 7 LFD) in 0.1M tris buffer at pH 9.0.The enzyme sample to be analyzed is dissolved in the same buffer. 10 mlsubstrate solution and 0.5 ml enzyme solution are mixed and transferredto a viscosimeter (e.g. Haake VT 181, NV sensor, 181 rpm), thermostatedat 40° C. Viscosity readings are taken as soon as possible after mixingand again 30 minutes later. The amount of enzyme that reduces theviscosity to one half under these conditions is defined as 1 unit ofCMC-endoase activity.

SDS polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectricfocusing with marker proteins in a manner known to persons skilled inthe art were used to determine the molecular weight and isolelectricpoint (pI), respectively, of the endoglucanase component in thecellulase preparation useful in the present context. In this way, themolecular weight of a specific endoglucanase component was determined tobe 43 kD. The isoelectric point of this endoglucanase was determined tobe about 5.1.

The cellobiohydrolase activity may be defined as the activity towardscellobiose p-nitrophenyl. The activity is determined as μmolenitrophenyl released per minute at 37° C. and pH 7.0. The presentendoglucanase component was found to have essentially nocellobiohydrolase activity.

The endoglucanase component in the cellulase preparation herein hasinitially been isolated by extensive purification procedures, i.a.involving reverse phase HPLC purification of a crude H. insolenscellulase mixture according to U.S. Pat. No. 4,435,307. This procedurehas surprisingly resulted in the isolation of a 43 kD endoglucanase as asingle component with unexpectedly favourable properties due to asurprisingly high endoglucanase activity.

Also, in addition to the screening test, the cellulase enzymes useful inthe present compositions can further be defined as enzymes exhibitingendoglucanase activity (in the following referred to as an"endoglucanase enzyme"), which enzymes have the amino acid sequenceshown in SEQ ID NO: 2, or a homologue thereof exhibiting endoglucanaseactivity.

In the present context, the term "homologue" is intended to indicate apolypeptide encoded by DNA which hybridizes to the same probe as the DNAcoding for the endoglucanase enzyme with this amino acid sequence undercertain specified conditions (such as presoaking in 5×SSC andprehybridizing for 1 h at 40° C. in a solution of 20% formamide,5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 ug ofdenatured sonicated calf thymus DNA, followed by hybridization in thesame solution supplemented with 100 μM ATP for 18 h at 40° C.). The termis intended to include derivatives of the aforementioned sequenceobtained by addition of one or more amino acid residues to either orboth the C- and N-terminal of the native sequence, substitution of oneor more amino acid residues at one or more sites in the native sequence,deletion of one or more amino acid residues at either or both ends ofthe native amino acid sequence or at one or more sites within the nativesequence, or insertion of one or more amino acid residues at one or moresites in the native sequence.

The endoglucanase enzyme herein may be one producible by species ofHumicola such as Humicola insolens e.g. strain DSM 1800, deposited onOct. 1, 1981 at the Deutsche Sammlung yon Mikroorganismen, MascheroderWeg 1B, D-3300 Braunschweig, FRG, in accordance with the provisions ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure (the BudapestTreaty).

In still a further aspect, the cellulase enzymes useful herein can bedefined, in addition to the screening test, as endoglucanase enzymeswhich have the amino acid sequence shown in SEQ ID NO: 4, or a homologuethereof (as defined above) exhibiting endoglucanase activity. Saidendoglucanase enzyme may be one producible by a species of Fusarium,such as Fusarium oxysporum, e.g. strain DSM 2672, deposited on Jun. 6,1983 at the Deutsche Sammlung von Mikroorganismen, Mascheroder Weg 1B,D-3300 Braunschweig, FRG, in accordance with the provisions of theBudapest Treaty.

Furthermore, it is contemplated that homologous endoglucanases may bederived from other microorganisms producing cellulolytic enzymes, e.g.species of Trichoderma, Myceliophthora, Phanerochaete, Schizophyllum,Penicillium, Aspergillus, and Geotricum.

For industrial production of the cellulase preparation herein, however,it is preferred to employ recombinant DNA techniques or other techniquesinvolving adjustements of fermentations or mutation of themicroorganisms involved to ensure overproduction of the desiredenzymatic activities. Such methods and techniques are known in the artand may readily be carried out by persons skilled in the art.

The endoglucanase component may thus be one which is producible by amethod comprising cultivating a host cell transformed with a recombinantDNA vector which carries a DNA sequence encoding said endoglucanasecomponent or a precursor of said endoglucanase component as well as DNAsequences encoding functions permitting the expression of the DNAsequence encoding the endoglucanase component or precursor thereof, in aculture medium under conditions permitting the expression of theendoglucanase component or precursor thereof and recovering theendoglucanase component from the culture.

DNA constructs comprising a DNA sequence encoding an endoglucanaseenzyme as described above, or a precursor form of the enzyme, includethe DNA constructs having a DNA sequence as shown in SEQ ID NO: 1 or SEQID. NO: 3, or a modification thereof. Examples of suitable mofidicationsof the DNA sequence are nucleotide substitutions which do not give riseto another amino acid sequence of the endoglucanase, but whichcorrespond to the codon usage of the host organism into which the DNAconstruct is introduced or nucleotide substitutions which do give riseto a different amino acid sequence and therefore, possibly, a differentprotein structure which might give rise to an endoglucanase mutant withdifferent properties than the native enzyme. Other examples of possiblemodifications are insertion of one or more nucleotides at either end ofthe sequence, or deletion of one or more nucleotides at either end orwithin the sequence.

DNA constructs encoding endoglucanase enzymes useful herein may beprepared synthetically by established standard methods, e.g. thephosphoamidite method described by S. L. Beaucage and M. H. Caruthers,Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described byMatthes et al., EMBO Journal 3, 1984, pp. 801-805. According to thephosphoamidite method, oligonucleotides are synthesized, e.g. in anautomatic DNA synthesizer, purified, annealed, ligated and cloned insuitable vectors.

A DNA construct encoding the endoglucanase enzyme or a precursor thereofmay, for instance, be isolated by establishing a cDNA or genomic libraryof a cellulase-producing microorganism, such as Humicola insolens, DSM1800, and screening for positive clones by conventional procedures suchas by hybridization using oligonucleotide probes synthesized on thebasis of the full or partial amino acid sequence of the endoglucanase inaccordance with standard techniques (cf. Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd. Ed. Cold Spring Harbor, 1989), or byselecting for clones expressing the appropriate enzyme activity (i.e.CMC-endoase activity as defined above), or by selecting for clonesproducing a protein which is reactive with an antibody against a nativecellulase (endoglucanase).

Finally, the DNA construct may be of mixed synthetic and genomic, mixedsynthetic and cDNA or mixed genomic and cDNA origin prepared by ligatingfragments of synthetic, genomic or cDNA origin (as appropriate), thefragments corresponding to various parts of the entire DNA construct, inaccordance with standard techniques. The DNA construct may also beprepared by polymerase chain reaction using specific primers, forinstance as described in U.S. Pat. No. 4,683,202 or R. K. Saiki et al.,Science 239, 1988, pp. 487-491.

Recombinant expression vectors into which the above DNA constructs areinserted include any vector which may conveniently be subjected torecombinant DNA procedures, and the choice of vector will often dependon the host cell into which it is to be introduced. Thus, the vector maybe an autonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowich it has been integrated.

In the vector, the DNA sequence encoding the endoglucanase should beoperably connected to a suitable promoter and terminator sequence. Thepromoter may be any DNA sequence which shows transcriptional activity inthe host cell of choice and may be derived from genes encoding proteinseither homologous or heterologous to the host cell. The procedures usedto ligate the DNA sequences coding for the endoglucanase, the promoterand the terminator, respectively, and to insert them into suitablevectors are well known to persons skilled in the art (cf., for instance,Sambrook et al., op.cit.).

Host cells which are transformed with the above DNA constructs or theabove expression vectors may be for instance belong to a species ofAspergillus, most preferably Aspergillys oryzae or Aspergillus niger.Fungal cells may be transformed by a process involving protoplastformation and transformation of the protoplasts followed by regenerationof the cell wall in a manner known per se. The use of Aspergillus as ahost microorganism is described in EP 238 023 (of Novo Industri A/S),the contents of which are hereby incorporated by reference. The hostcell may also be a yeast cell, e.g. a strain of Saccharomycescerevisiae.

Alternatively, the host organism may be a bacterium, in particularstrains of Streptomyces and Bacillus, and E. coli. The transformation ofbacterial cells may be performed according to conventional methods, e.g.as described in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, 1989.

The screening of appropriate DNA sequences and construction of vectorsmay also be carried out by standard procedures, cf. Sambrook et al.,op.cot.

The medium used to cultivate the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed endoglucanase may conveniently be secreted into the culturemedium and may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

By employing recombinant DNA techniques as indicated above, techniquesof protein purification, techniques of fermentation and mutation orother techniques which are well known in the art, it is possible toprovide endoglucanases of a high purity.

The level in the present composition of cellulase described above shouldbe such that the amount of enzyme protein to be delivered in the washsolution is from 0.005 to 40 mg/liter of wash solution, preferably 0.01to 10 mg/liter of wash solution.

Peroxidases

The peroxidases which may be employed for the present purpose may beisolated from and are producible by plants (e.g. horseradish peroxidase)or micororganisms such as fungi or bacteria. Some preferred fungiinclude strains belonging to the subdivision Deuteromycotina, classHyphomycetes, e.g. Fusarium, Humicola, Tricoderma, Myrothecium,Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia,Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672),Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO 6113),Verticilluum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERMP-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alliorDreschlera halodes.

Other preferred fungi inlclude strains belonging to the subdivisionBasidiomycotina, class Basidiomycetes, e.g. Coprinus, Phanerochaete,Coriolus or Trametes, in particular Coprinus cinereus f. microsporus(IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g.NA-12) or Coriolus versicolor (e.g. PR4 28-A).

Further preferred fungi include strains belonging to the subdivisionZygomycotina, class Mycoraceae, e.g. Rhizopus or Mucor, in particularMucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales,e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus(IFO 12382) or Streptoverticillum verticillium ssp. verticillium.

Other preferred bacteria inlude Bacillus pumillus (ATCC 12905), Bacillusstearothermophilus, Rhododbacter sphaeroides, Rhodomonas palustri,Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) or Pseudomonasfluorescens (NRRL B-11).

Other potential sources of useful peroxidases are listed in B. C.Saunders et al., op. cit., pp. 41-43.

Methods of producing enzymes to be used according to the invention aredescribed in the art, cf. for example FEBS Letters 1625, 173(1), Appliedand Environmental Microbiology, February 1985, pp. 273-278, AppliedMicrobiol. Biotechnol. 26, 1987, pp. 158-163, Biotechnology Letters9(5), 1987, pp. 357-360, Nature 326, 2 Apr. 1987, FEBS Letters 4270,209(2), p. 321, EP 179 486, EP 200 565, GB 2 167 421, EP 171 074, andAgric. Biol. Chem. 50(1), 1986, p. 247.

Particularly preferred peroxidases are those which are active at thetypical pH of washing liquors, i.e. at a pH of 6.5-10.5, preferably6.5-9.5, and most preferably 7.5-9.5. Such enzymes may be isolated byscreening for the relevant enzyme production by alkalophilicmicroorganisms, e.g. using the ABTS assay described in R. E. Childs andW. G. Bardsley, Biochem. J. 145, 1975, pp. 93-103.

Other preferred peroxidases are those which exhibit a goodthermostability as well as a good stability towards commonly useddetergent components such as non-ionic, cationic, or anionicsurfactants, detergent builders, phosphate etc.

Another group of useful peroxidases are haloperoxidases, such as chloro-and bromoperoxidases.

The peroxidase-enzyme may futhermore be one which is producible by amethod comprising cultivating a host cell transformed with a recombinantDNA vector which carries a DNA sequence encoding said enzyme as well asDNA sequences encoding functions permitting the expression of the DNAsequence encoding the enzyme, in a culture medium under conditionspermitting the expression of the enzyme and recovering the enzyme fromthe culture.

A DNA fragment encoding the enzyme may, for instance, be isolated byestablishing a cDNA or genomic library of a microorganism producing theenzyme of interest, such as one of the organisms mentioned above, andscreening for positive clones by conventional procedures such as byhybridization to oligonucleotide probes synthesized on the basis of thefull or partial amino acid sequence of the enzyme, or by selecting forclones expressing the appropriate enzyme activity, or by selecting forclones producing a protein which is reactive with an antibody againstthe native enzyme.

Once selected, the DNA sequence may be inserted into a suitablereplicable expression vector comprising appropriate promotor, operatorand terminator sequences permitting the enzyme to be expressed in aparticular host organism, as well as an origin of replication, enablingthe vector to replicate in the host organism in question.

The resulting expression vector may then be transformed into a suitablehost cell, such as a fungal cell, preferred examples of which are aspecies of Aspergillus, most preferably Aspergillus oryzae orAspergillus niger. Fungal cells may be transformed by a processinvolving protoplast formation and transformation of the protoplastsfollowed by regeneration of the cell wall in a manner known per se. Theuse of Aspergillus as a host micororganism is described in EP 238,023(of Novo Industri A/S), the contents of which are hereby incorporated byreference.

Alternatively, the host organisms may be a bacterium, in particularstrains of Streptomyces and Bacillus, or E. coli. The transformation ofbacterial cells may be performed according to conventional methods, e.g.as described in T. Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, 1982.

The screening of appropriate DNA sequences and construction of vectorsmay also be carried out by standard procedures, cf. T. Maniatis et al.,op. cit.

The medium used to cultivate the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed enzyme may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

The screening of appropriate DNA sequences and construction of vectorsmay also be carried out by standard procedures, cf. T. Maniatis et al.,op. cit.

The medium used to cultivate the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed enzyme may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

At the beginning or during the process, H₂ O₂ may be added, e.g. in anamount of 0.001-5 mM, particularly 0.01-1 mM. When using Coprinusperoxidase, 0.01-0.25 mM H₂ O₂ is preferred, and with B. pumilusperoxidase 0.1-1 mM H₂ O₂.

The hydrogen peroxide may be added as hydrogen peroxide or a precursorthereof, preferably a perborate or percarbonate. The level of hydrogenperoxide precursor that can be used is dependent on the specificproperties of the peroxidase chosen, e.g. Coprinus peroxidase should beapplied in a detergent composition which contains less than 5%perborate.

In the process of this invention, it may be desirable to utilize anenzymatic process for hydrogen peroxide formation. Thus, the processaccording to the invention may additionally comprise adding an enzymaticsystem (i.e. an enzyme and a substrate therefor) which is capable ofgenerating hydrogen peroxide at the beginning or during the washingand/or rinsing process.

One such category of hydrogen peroxide generating systems comprisesenzymes which are able to convert molecular oxygen and an organic orinorganic substrate into hydrogen peroxide and the oxidized substraterespectively. These enzymes produce only low levels of hydrogenperoxide, but they may be employed to great advantage in the process ofthe invention as the presence of peroxidase ensures an efficientutilization of the hydrogen peroxide produced.

Preferred hydrogen peroxide-generating enzymes are those which act oncheap and readily available substrates which may conveniently beincluded into detergent compositions. An example of such a substrate isglucose which may be utilized for hydrogen peroxide production by meansof glucose oxidase. Suitable oxidases include those which act onaromatic compounds such as phenols and related substances, e.g. catecholoxidases, laccase. Other suitable oxidases are urate oxidase, galactoseoxidase, alcohol oxidases, amine oxidases, amino acid oxidase,amyloglucosidase, and cholesterol oxidase.

The preferred enzymatic systems are alcohol and aldehyde oxidases.

The more preferred systems for granular detergent application would havesolid alcohols, e.g. glucose whose oxidation is catalysed by glucoseoxidase to glucoronic acid with the formation of hydrogen peroxide.

The more preferred systems for liquid detergent application wouldinvolve liquid alcohols which could also act as, for example, solvents.An example is ethanol/ethanol oxidase.

The quantity of oxidase to be employed in compositions according to theinvention should be at least sufficient to provide a constant generationof 0.01 to 10 ppm AvO per minute in the wash. For example, with theglucose oxidase, this can be achieved at room temperature and at pH 6 to11, preferentially 7 to 9 with 50-5000 U/l glucose oxidase, 0.005 to0.5% glucose under constant aeration.

The addition of another oxidisable substrate for the peroxidase at thebeginning or during the washing and/or rinsing process may enhance thedye transfer inhibitory effect of the peroxidase employed. This isthought to be ascribable to the formation of short-lived radicals orother oxidised states of this substrate which participate in thebleaching or other modification of the coloured substance. Examples ofsuch oxidisable substrates are metal ions, e.g. Mn⁺⁺, halide ions, e.g.chloride or bromide ions, or organic compounds such as phenols, e.g.p-hydroxycinnamic acid or 2,4-dichlorophenol. Other examples of phenoliccompounds which may be used for the present purpose are those given inM. Kato and S. Shimizu, Plant Cell Physiol. 26(7), 1985, pp. 1291-1301(cf. Table 1 in particular) or B. C. Saunders et al., op. cit., p. 141ff. The amount of oxidisable substrate to be added is suitably betweenabout 1 μM and 1 mM.

In the process of the invention, the peroxidase will typically be addedas a component of a detergent composition and may be added in an amountof 0.01-100 mg enzyme per liter of wash liquid. As such, it may beincluded in the detergent composition in the form of a non-dustinggranulate, a liquid, in particular a stabilized liquid, or a protectedenzyme. Non-dusting granulates may be produced, e.g. as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) andmay optionally be coated by methods known in the art. Liquid enzymepreparations may, for instance, be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acidaccording to established methods. Other enzyme stabilizers are wellknown in the art. Protected enzymes may be prepared according to themethod disclosed in EP 238,216. The detergent composition may alsocomprise one or more substrates for the peroxidase. Usually, the pH of asolution of the detergent composition of the invention will bepreferably from 7-12, especially from 7.5 to 9.5. The wash pH isdependent on the peroxidase chosen, e.g. Coprinus peroxidase should beapplied in a wash pH below 9.5.

Detergent Adjuncts

The composition of the present can contain the usual components of suchdetergent compositions in the usual amounts. Thus, organic surfactantsanionic, nonionic, ampholytic, or zwitterionic or less usually cationicand mixtures thereof, may be present. Suitable surfactants are wellknown in the art and an extensive list of such compounds is given inU.S. Pat. No. 3,717,630 and in U.S. patent application Ser. No. 589,116.

Detergent compositions useful in the present invention contain from 1 to95%, preferable from 5 to 40% of a nonionic, anionic, zwitterionic, ormixtures thereof. Detergency builders, whether inorganic or organic,phosphatic or not, water-soluble or insoluble, and other water-solublesalts may be present, and salts of this sort may be employed whetherorganic detergents are present or not. A description of suitablebuilders is given in U.S. Pat. No. 3,936,537 and in U.S. patentapplication Ser. No. 589,116. Detergent builders are present from 0 to50%, preferably from 5 to 40%.

Other components used in detergent compositions may be employed, such assuds boosting or depressing agents, enzymes and stabilizers oractivators, soil-suspending agents soil-release agents, opticalbrighteners, abrasives, bactericides, tarnish inhibitors, coloringagents, and perfumes.

These components, particularly the enzymes, optical brighteners,coloring agents, and perfumes, should preferably be chosen such thatthey are compatible with the bleach component of the composition.

The detergent compositions according to the invention can be in liquid,paste or granular forms. The enzyme may be formulated in any convenientform, e.g. as a powder or liquid. The enzyme may be stabilized in aliquid by inclusion of enzyme stabilizers. Liquid deterents may furtherinclude stabilized hydrogen peroxide precursors. Granular compositionsaccording to the present invention can also be in "compact form", i.e.they may have a relatively higher density than conventional granulardetergents, i.e. from 550 to 950 g/l; in such case, the granulardetergent compositions according to the present invention will contain alower amount of "inorganic filler salt", compared to conventionalgranular detergents; typical filler salts are alkaline earth metal saltsof sulphates and chlorides, typically sodium sulphate; "compact"detergents typically comprise not more than 10% filler salt.

The present invention also relates to a process for inhibiting dyetransfer from one fabric to another of solubilized and suspended dyesencountered during fabric laundering operations involving coloredfabrics.

The process comprises contacting fabrics with a laundering solution ashereinbefore described.

The process of the invention is conveniently carried out in the courseof the washing process. The washing process is preferably carried out at5° C. to 75° C., especially 20° C. to 60° C. The pH of the treatmentsolution is preferably from 7 to 12, especially from 7 to 9.5.

The process and compositions of the invention can also be used asadditive during laundry operations.

The following examples illustrate the present invention and theunexpected superior colour care benefits obtained therefrom.

EXAMPLE I Criticality of the Cellulase Performance Parameter of Claim 1

The following test was conducted:

Test Conditions

washing temperature: 60° C. (heat up cycle)

Washing time: 40 min.

pH=7.5

Water hardness: 4 mmol/L

Detergent concentration: 1%

Detergent composition: crf. EPA 350 098 ex. 1

Cellulases:

1) Celluzyme® supplied by Novo Nordisk=reference

2) 43 kD endoglucanase=cellulase according to the invention

Test Results

    ______________________________________                                                            % C14-CMC                                                                     Removal by Cellulase                                      ______________________________________                                        Detergent without cellulase (= reference)                                                           0                                                       Detergent + Celluzyme ®                                                   1.5 mg protein/L (150 × 10.sup.-6 %)                                                          12.7                                                    3.0 mg protein/L (300 × 10.sup.-6 %)                                                          17.7                                                    4.5 mg protein/L (450 × 10.sup.-6 %)                                                          21.5                                                    Detergent + 43 kD endoglucanase                                                                     20.3                                                    0.3 mg protein/L (30 × 10.sup.-6 %)                                     ______________________________________                                    

Discussion of the Results

The above data clearly demonstrate the criticality of the claimedparameter for the cellulases of the invention over the commerciallyavailable Celluzyme.

EXAMPLE II

Two sets of each four types of detergent compositions are prepared, allbased on a compact granular.

Such a compact granular detergent composition typically contains thefollowing ingredients:

    ______________________________________                                        Linear alkyl benzene sulphonate (LAS)                                                                   11%                                                 Alkyl suphate              5%                                                 Nonionic                   6%                                                 Trisodium citrate         15%                                                 Zeolite                   32%                                                 Citric acid                6%                                                 Polymer                    4%                                                 Chelant                    0.2%                                               Sodium sulphate            5%                                                 Sodium silicate            2%                                                 Perborate                  0.5%                                               Phenol sulphonate          0.1%                                               ______________________________________                                    

The above detergent composition was supplemented as indicated in below:##STR1## A) without 43 kD endoglucanase and peroxidase=reference B) with43 kD endoglucanase

C) with peroxide

D) with 43 kD endoglucanase and peroxidase ##STR2## A) withoutcelluzyme® and peroxidase B) with celluzyme®

C) with peroxidase

D) with celluzyme® and peroxidase

The first type of each set of detergent compositions does not containany cellulase and peroxidase (reference composition: A).

In the first set of detergent compositions the 43 kD endoglucanase isadded at a level of 2 mg enzyme protein/liter of the wash solution (55cevu/liter). In the second set of the detergent compositions thecelluzyme® is added at a level of 76 mg enzyme protein/liter of the washsolution (55 cevu/liter).

Test Conditions

Test in Miele washing machine

Cotton program, low water level (181), short cycle

4 cycles

Temperature 40° C.

Composition of the Wash-load

1) 1 kg clean load:

150 g cotton (terry)

150 g knitted cotton (underwear)

200 g woven cotton

300 g PE/cotton

200 g PE

2) For whiteness grading: 3 white, soiled, items (4 replicates each).

3) 10 by 5 cm acid red 151 dye on nylon: To create a low dye transferlevel.

4) Prepared stains to provide a source of typical laundry soil.

Hard water (15 grs/glln).

Detergent concentration=0.6%.

Test Procedure

The design of the test is such as to compare whiteness of the textileitems, laundered cumulatively 4 times between the compositions to betested and the reference composition. Three soiled items were used forthis test. For each treatment of an item four replicates were used. Theitems to be examined are displayed on a flat, neutral colored gradingsurface parallel to the light source. As a light source a fluorescentlight is used: 27 Philips "cold" color TL 40/57 producing 1080 WATTS oflight designed to match with regular daylight (D65). The color T° is of7400° K., the color reflectance is excellent (94) and the light outputis 46 LM/W.

Differences are recorded in panel score units (psu), positive beingperformancewise better than the reference treatment.

Grading Scale (PSU Grading)

0=Equal

1=I think this one is better

2=I know this one is a little better

3=This one is a lot better

4=This one is a whole lot better

The PSU grading data are statistical recount, an average of the 4replicates is made, LSD (least significant difference) is mentioned intable I and II.

                  TABLE III                                                       ______________________________________                                        Test results: peroxidase/celluzyme ®                                               B vs. A                                                                              C vs. A    D vs. A  LSD                                       ______________________________________                                        2 cycles AV                                                                              0.28     1.67 s     1.80 s 0.61                                    3 cycles AV                                                                              0.22     2.02 s     2.26 s 0.50                                    4 cycles AV                                                                              0.68     2.48 s     2.66 s 0.76                                    ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Test results: peroxidase/43 kD endoglucanase                                           B vs. A                                                                              C vs. A    D vs. A  LSD                                       ______________________________________                                        2 cycles AV                                                                              -0.10    1.38 s     1.97 s 0.40                                    3 cycles AV                                                                              -0.30    2.25 s     2.51 s 0.46                                    4 cycles AV                                                                              0.07     2.44 s     2.97 s 0.29                                    ______________________________________                                    

Conclusion

The above results clearly show that the peroxidase/43 kD combination ofthe present invention gives a statistically significant betterperformance than the sum of the individual actions of both ingredients.

EXAMPLE III to VIII

The following compositions are made.

a) Compact granular detergent: examples II to IV.

    ______________________________________                                                             EXAMPLES                                                                      III  IV                                                  ______________________________________                                        Linear alkyl benzene sulphonate                                                                      11.4   10.70                                           Tallow alkyl sulphate  1.80   2.40                                            C.sub.45 alkyl sulphate                                                                              3.00   3.10                                            C.sub.45 alcohol 7 times ethoxylated                                                                 4.00   4.00                                            Tallow alcohol 11 times ethoxylated                                                                  1.80   1.80                                            Dispersant             0.07   0.1                                             Silicone fluid         0.80   0.80                                            Trisodium citrate      14.00  15.00                                           Citric acid            3.00   2.50                                            Zeolite                32.50  32.10                                           Maleic acid acrylic acid copolymer                                                                   5.00   5.00                                            DETMPA                 1.00   0.20                                            Cellulase 43 kD endoglucanase                                                                        0.03   0.025                                           Alkalase               0.60   0.60                                            Lipase                 0.36   0.40                                            Amylase                0.30   0.30                                            Sodium silicate        2.00   2.50                                            Sodium sulphate        3.50   5.20                                            PVP                    0.30   0.50                                            Perborate              0.5    1                                               Phenol sulphonate      0.1    0.2                                             Peroxidase             0.1    0.1                                             Minors                 up to 100                                              ______________________________________                                    

b) conventional granular detergent: examples V and VI

    ______________________________________                                                               EXAMPLES                                                                      V    IV                                                ______________________________________                                        Sodium linear C.sub.12 alkyl benzene sulfonate                                                         6.5    8.0                                           Sodium sulfate           15.0   18.0                                          Zeolite A                26.0   22.0                                          Sodium nitrilotriacetate 5.0    5.0                                           Cellulase 43 kD endoglucanase                                                                          0.02   0.03                                          PVP                      0.5    0.7                                           TAED                     3.0    3.0                                           Boric acid               4.0    --                                            Perborate                0.5    1                                             Phenol sulphonate        0.1    0.2                                           Peroxidase               0.1    0.2                                           Minors                   up to 100                                            ______________________________________                                    

c) liquid detergent: examples VII and VIII

    ______________________________________                                                             EXAMPLES                                                                      VII  VIII                                                ______________________________________                                        C.sub.12-14 alkenyl succinic acid                                                                    3.0    8.0                                             Citric acid monohydrate                                                                              10.0   15.0                                            Sodium C.sub.12-15 alkyl sulphate                                                                    8.0    8.0                                             Sodium sulfate of C12-15 alcohol 2                                                                   --     3.0                                             times ethoxylated                                                             C.sub.12-15 alcohol 7 times ethoxylated                                                              --     8.0                                             C.sub.12-15 alcohol 5 times ethoxylated                                                              8.0    --                                              Diethylene triamine penta (methylene                                                                 0.2    --                                              phosphonic acid)                                                              Oleic acid             1.8    --                                              Ethanol                4.0    4.0                                             Propanediol            2.0    2.0                                             Protease               0.2    0.2                                             Cellulase 43 kD endoglucanase                                                                        0.2    0.05                                            PVP                    1.0    2.0                                             Suds suppressor        0.15   0.15                                            NaOH                   up to pH 7.5                                           Perborate              0.5    1                                               Phenol sulphonate      0.1    0.2                                             Peroxidase             0.4    0.1                                             Waters and minors      up to 100 parts                                        ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1060 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 10..924                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGATCCAAGATGCGTTCCTCCCCCCTCCTCCCGTCCGCCGTTGTGGCC48                            MetArgSerSerProLeuLeuProSerAlaValValAla                                       1510                                                                          GCCCTGCCGGTGTTGGCCCTTGCCGCTGATGGCAGGTCCACCCGCTAC96                            AlaLeuProValLeuAlaLeuAlaAlaAspGlyArgSerThrArgTyr                              152025                                                                        TGGGACTGCTGCAAGCCTTCGTGCGGCTGGGCCAAGAAGGCTCCCGTG144                           TrpAspCysCysLysProSerCysGlyTrpAlaLysLysAlaProVal                              30354045                                                                      AACCAGCCTGTCTTTTCCTGCAACGCCAACTTCCAGCGTATCACGGAC192                           AsnGlnProValPheSerCysAsnAlaAsnPheGlnArgIleThrAsp                              505560                                                                        TTCGACGCCAAGTCCGGCTGCGAGCCGGGCGGTGTCGCCTACTCGTGC240                           PheAspAlaLysSerGlyCysGluProGlyGlyValAlaTyrSerCys                              657075                                                                        GCCGACCAGACCCCATGGGCTGTGAACGACGACTTCGCGCTCGGTTTT288                           AlaAspGlnThrProTrpAlaValAsnAspAspPheAlaLeuGlyPhe                              808590                                                                        GCTGCCACCTCTATTGCCGGCAGCAATGAGGCGGGCTGGTGCTGCGCC336                           AlaAlaThrSerIleAlaGlySerAsnGluAlaGlyTrpCysCysAla                              95100105                                                                      TGCTACGAGCTCACCTTCACATCCGGTCCTGTTGCTGGCAAGAAGATG384                           CysTyrGluLeuThrPheThrSerGlyProValAlaGlyLysLysMet                              110115120125                                                                  GTCGTCCAGTCCACCAGCACTGGCGGTGATCTTGGCAGCAACCACTTC432                           ValValGlnSerThrSerThrGlyGlyAspLeuGlySerAsnHisPhe                              130135140                                                                     GATCTCAACATCCCCGGCGGCGGCGTCGGCATCTTCGACGGATGCACT480                           AspLeuAsnIleProGlyGlyGlyValGlyIlePheAspGlyCysThr                              145150155                                                                     CCCCAGTTCGGCGGTCTGCCCGGCCAGCGCTACGGCGGCATCTCGTCC528                           ProGlnPheGlyGlyLeuProGlyGlnArgTyrGlyGlyIleSerSer                              160165170                                                                     CGCAACGAGTGCGATCGGTTCCCCGACGCCCTCAAGCCCGGCTGCTAC576                           ArgAsnGluCysAspArgPheProAspAlaLeuLysProGlyCysTyr                              175180185                                                                     TGGCGCTTCGACTGGTTCAAGAACGCCGACAATCCGAGCTTCAGCTTC624                           TrpArgPheAspTrpPheLysAsnAlaAspAsnProSerPheSerPhe                              190195200205                                                                  CGTCAGGTCCAGTGCCCAGCCGAGCTCGTCGCTCGCACCGGATGCCGC672                           ArgGlnValGlnCysProAlaGluLeuValAlaArgThrGlyCysArg                              210215220                                                                     CGCAACGACGACGGCAACTTCCCTGCCGTCCAGATCCCCTCCAGCAGC720                           ArgAsnAspAspGlyAsnPheProAlaValGlnIleProSerSerSer                              225230235                                                                     ACCAGCTCTCCGGTCAACCAGCCTACCAGCACCAGCACCACGTCCACC768                           ThrSerSerProValAsnGlnProThrSerThrSerThrThrSerThr                              240245250                                                                     TCCACCACCTCGAGCCCGCCAGTCCAGCCTACGACTCCCAGCGGCTGC816                           SerThrThrSerSerProProValGlnProThrThrProSerGlyCys                              255260265                                                                     ACTGCTGAGAGGTGGGCTCAGTGCGGCGGCAATGGCTGGAGCGGCTGC864                           ThrAlaGluArgTrpAlaGlnCysGlyGlyAsnGlyTrpSerGlyCys                              270275280285                                                                  ACCACCTGCGTCGCTGGCAGCACTTGCACGAAGATTAATGACTGGTAC912                           ThrThrCysValAlaGlySerThrCysThrLysIleAsnAspTrpTyr                              290295300                                                                     CATCAGTGCCTGTAGACGCAGGGCAGCTTGAGGGCCTTACTGGTGGCCGCAA964                       HisGlnCysLeu                                                                  305                                                                           CGAAATGACACTCCCAATCACTGTATTAGTTCTTGTACATAATTTCGTCATCCCTCCAGG1024              GATTGTCACATAAATGCAATGAGGAACAATGAGTAC1060                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 305 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetArgSerSerProLeuLeuProSerAlaValValAlaAlaLeuPro                              151015                                                                        ValLeuAlaLeuAlaAlaAspGlyArgSerThrArgTyrTrpAspCys                              202530                                                                        CysLysProSerCysGlyTrpAlaLysLysAlaProValAsnGlnPro                              354045                                                                        ValPheSerCysAsnAlaAsnPheGlnArgIleThrAspPheAspAla                              505560                                                                        LysSerGlyCysGluProGlyGlyValAlaTyrSerCysAlaAspGln                              65707580                                                                      ThrProTrpAlaValAsnAspAspPheAlaLeuGlyPheAlaAlaThr                              859095                                                                        SerIleAlaGlySerAsnGluAlaGlyTrpCysCysAlaCysTyrGlu                              100105110                                                                     LeuThrPheThrSerGlyProValAlaGlyLysLysMetValValGln                              115120125                                                                     SerThrSerThrGlyGlyAspLeuGlySerAsnHisPheAspLeuAsn                              130135140                                                                     IleProGlyGlyGlyValGlyIlePheAspGlyCysThrProGlnPhe                              145150155160                                                                  GlyGlyLeuProGlyGlnArgTyrGlyGlyIleSerSerArgAsnGlu                              165170175                                                                     CysAspArgPheProAspAlaLeuLysProGlyCysTyrTrpArgPhe                              180185190                                                                     AspTrpPheLysAsnAlaAspAsnProSerPheSerPheArgGlnVal                              195200205                                                                     GlnCysProAlaGluLeuValAlaArgThrGlyCysArgArgAsnAsp                              210215220                                                                     AspGlyAsnPheProAlaValGlnIleProSerSerSerThrSerSer                              225230235240                                                                  ProValAsnGlnProThrSerThrSerThrThrSerThrSerThrThr                              245250255                                                                     SerSerProProValGlnProThrThrProSerGlyCysThrAlaGlu                              260265270                                                                     ArgTrpAlaGlnCysGlyGlyAsnGlyTrpSerGlyCysThrThrCys                              275280285                                                                     ValAlaGlySerThrCysThrLysIleAsnAspTrpTyrHisGlnCys                              290295300                                                                     Leu                                                                           305                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1473 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 97..1224                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAATTCGCGGCCGCTCATTCACTTCATTCATTCTTTAGAATTACATACACTCTCTTTCAA60                AACAGTCACTCTTTAAACAAAACAACTTTTGCAACAATGCGATCTTACACTCTT114                     MetArgSerTyrThrLeu                                                            15                                                                            CTCGCCCTGGCCGGCCCTCTCGCCGTGAGTGCTGCTTCTGGAAGCGGT162                           LeuAlaLeuAlaGlyProLeuAlaValSerAlaAlaSerGlySerGly                              101520                                                                        CACTCTACTCGATACTGGGATTGCTGCAAGCCTTCTTGCTCTTGGAGC210                           HisSerThrArgTyrTrpAspCysCysLysProSerCysSerTrpSer                              253035                                                                        GGAAAGGCTGCTGTCAACGCCCCTGCTTTAACTTGTGATAAGAACGAC258                           GlyLysAlaAlaValAsnAlaProAlaLeuThrCysAspLysAsnAsp                              404550                                                                        AACCCCATTTCCAACACCAATGCTGTCAACGGTTGTGAGGGTGGTGGT306                           AsnProIleSerAsnThrAsnAlaValAsnGlyCysGluGlyGlyGly                              55606570                                                                      TCTGCTTATGCTTGCACCAACTACTCTCCCTGGGCTGTCAACGATGAG354                           SerAlaTyrAlaCysThrAsnTyrSerProTrpAlaValAsnAspGlu                              758085                                                                        CTTGCCTACGGTTTCGCTGCTACCAAGATCTCCGGTGGCTCCGAGGCC402                           LeuAlaTyrGlyPheAlaAlaThrLysIleSerGlyGlySerGluAla                              9095100                                                                       AGCTGGTGCTGTGCTTGCTATGCTTTGACCTTCACCACTGGCCCCGTC450                           SerTrpCysCysAlaCysTyrAlaLeuThrPheThrThrGlyProVal                              105110115                                                                     AAGGGCAAGAAGATGATCGTCCAGTCCACCAACACTGGAGGTGATCTC498                           LysGlyLysLysMetIleValGlnSerThrAsnThrGlyGlyAspLeu                              120125130                                                                     GGCGACAACCACTTCGATCTCATGATGCCCGGCGGTGGTGTCGGTATC546                           GlyAspAsnHisPheAspLeuMetMetProGlyGlyGlyValGlyIle                              135140145150                                                                  TTCGACGGCTGCACCTCTGAGTTCGGCAAGGCTCTCGGCGGTGCCCAG594                           PheAspGlyCysThrSerGluPheGlyLysAlaLeuGlyGlyAlaGln                              155160165                                                                     TACGGCGGTATCTCCTCCCGAAGCGAATGTGATAGCTACCCCGAGCTT642                           TyrGlyGlyIleSerSerArgSerGluCysAspSerTyrProGluLeu                              170175180                                                                     CTCAAGGACGGTTGCCACTGGCGATTCGACTGGTTCGAGAACGCCGAC690                           LeuLysAspGlyCysHisTrpArgPheAspTrpPheGluAsnAlaAsp                              185190195                                                                     AACCCTGACTTCACCTTTGAGCAGGTTCAGTGCCCCAAGGCTCTCCTC738                           AsnProAspPheThrPheGluGlnValGlnCysProLysAlaLeuLeu                              200205210                                                                     GACATCAGTGGATGCAAGCGTGATGACGACTCCAGCTTCCCTGCCTTC786                           AspIleSerGlyCysLysArgAspAspAspSerSerPheProAlaPhe                              215220225230                                                                  AAGGTTGATACCTCGGCCAGCAAGCCCCAGCCCTCCAGCTCCGCTAAG834                           LysValAspThrSerAlaSerLysProGlnProSerSerSerAlaLys                              235240245                                                                     AAGACCACCTCCGCTGCTGCTGCCGCTCAGCCCCAGAAGACCAAGGAT882                           LysThrThrSerAlaAlaAlaAlaAlaGlnProGlnLysThrLysAsp                              250255260                                                                     TCCGCTCCTGTTGTCCAGAAGTCCTCCACCAAGCCTGCCGCTCAGCCC930                           SerAlaProValValGlnLysSerSerThrLysProAlaAlaGlnPro                              265270275                                                                     GAGCCTACTAAGCCCGCCGACAAGCCCCAGACCGACAAGCCTGTCGCC978                           GluProThrLysProAlaAspLysProGlnThrAspLysProValAla                              280285290                                                                     ACCAAGCCTGCTGCTACCAAGCCCGTCCAACCTGTCAACAAGCCCAAG1026                          ThrLysProAlaAlaThrLysProValGlnProValAsnLysProLys                              295300305310                                                                  ACAACCCAGAAGGTCCGTGGAACCAAAACCCGAGGAAGCTGCCCGGCC1074                          ThrThrGlnLysValArgGlyThrLysThrArgGlySerCysProAla                              315320325                                                                     AAGACTGACGCTACCGCCAAGGCCTCCGTTGTCCCTGCTTATTACCAG1122                          LysThrAspAlaThrAlaLysAlaSerValValProAlaTyrTyrGln                              330335340                                                                     TGTGGTGGTTCCAAGTCCGCTTATCCCAACGGCAACCTCGCTTGCGCT1170                          CysGlyGlySerLysSerAlaTyrProAsnGlyAsnLeuAlaCysAla                              345350355                                                                     ACTGGAAGCAAGTGTGTCAAGCAGAACGAGTACTACTCCCAGTGTGTC1218                          ThrGlySerLysCysValLysGlnAsnGluTyrTyrSerGlnCysVal                              360365370                                                                     CCCAACTAAATGGTAGATCCATCGGTTGTGGAAGAGACTATGCGTCTCAGAAGGGA1274                  ProAsn                                                                        375                                                                           TCCTCTCATGAGCAGGCTTGTCATTGTATAGCATGGCATCCTGGACCAAGTGTTCGACCC1334              TTGTTGTACATAGTATATCTTCATTGTATATATTTAGACACATAGATAGCCTCTTGTCAG1394              CGACAACTGGCTACAAAAGACTTGGCAGGCTTGTTCAATATTGACACAGTTTCCTCCATA1454              AAAAAAAAAAAAAAAAAAA1473                                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 376 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetArgSerTyrThrLeuLeuAlaLeuAlaGlyProLeuAlaValSer                              151015                                                                        AlaAlaSerGlySerGlyHisSerThrArgTyrTrpAspCysCysLys                              202530                                                                        ProSerCysSerTrpSerGlyLysAlaAlaValAsnAlaProAlaLeu                              354045                                                                        ThrCysAspLysAsnAspAsnProIleSerAsnThrAsnAlaValAsn                              505560                                                                        GlyCysGluGlyGlyGlySerAlaTyrAlaCysThrAsnTyrSerPro                              65707580                                                                      TrpAlaValAsnAspGluLeuAlaTyrGlyPheAlaAlaThrLysIle                              859095                                                                        SerGlyGlySerGluAlaSerTrpCysCysAlaCysTyrAlaLeuThr                              100105110                                                                     PheThrThrGlyProValLysGlyLysLysMetIleValGlnSerThr                              115120125                                                                     AsnThrGlyGlyAspLeuGlyAspAsnHisPheAspLeuMetMetPro                              130135140                                                                     GlyGlyGlyValGlyIlePheAspGlyCysThrSerGluPheGlyLys                              145150155160                                                                  AlaLeuGlyGlyAlaGlnTyrGlyGlyIleSerSerArgSerGluCys                              165170175                                                                     AspSerTyrProGluLeuLeuLysAspGlyCysHisTrpArgPheAsp                              180185190                                                                     TrpPheGluAsnAlaAspAsnProAspPheThrPheGluGlnValGln                              195200205                                                                     CysProLysAlaLeuLeuAspIleSerGlyCysLysArgAspAspAsp                              210215220                                                                     SerSerPheProAlaPheLysValAspThrSerAlaSerLysProGln                              225230235240                                                                  ProSerSerSerAlaLysLysThrThrSerAlaAlaAlaAlaAlaGln                              245250255                                                                     ProGlnLysThrLysAspSerAlaProValValGlnLysSerSerThr                              260265270                                                                     LysProAlaAlaGlnProGluProThrLysProAlaAspLysProGln                              275280285                                                                     ThrAspLysProValAlaThrLysProAlaAlaThrLysProValGln                              290295300                                                                     ProValAsnLysProLysThrThrGlnLysValArgGlyThrLysThr                              305310315320                                                                  ArgGlySerCysProAlaLysThrAspAlaThrAlaLysAlaSerVal                              325330335                                                                     ValProAlaTyrTyrGlnCysGlyGlySerLysSerAlaTyrProAsn                              340345350                                                                     GlyAsnLeuAlaCysAlaThrGlySerLysCysValLysGlnAsnGlu                              355360365                                                                     TyrTyrSerGlnCysValProAsn                                                      370375                                              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What is claimed is:
 1. A dye transfer inhibiting composition comprisingan enzyme exhibiting peroxidase activity, a hydrogen peroxide or a hydrogen peroxide precursor compound, or an enzymatic system capable of generating hydrogen peroxide, an oxidizable substrate for said enzyme exhibiting peroxidase activity, and a cellulase, wherein said cellulase has the amino acid sequence shown in the appended sequence listing ID#2, which provides at least 10% removal of immobilized radioactive labeled carboxymethyl cellulose according to the C14CMC method at 25×10⁻⁶ % by weight of the cellulase protein in the test solution.
 2. A dye transfer inhibiting composition according to claim 1 wherein the cellulase consists essentially of a homogeneous endoglucanase component which is immunoreactive with an antibody raised against a highly purified about 43 kD cellulase derived from Humicola insolens, DSM 1800, or which is homologous to said 43 kD endoglucanase.
 3. A dye transfer inhibiting composition according to claim 2 wherein the endoglucanase component of said cellulase has an isoelectric point of about 5.1.
 4. A dye transfer inhibiting composition according to claim 2 wherein the composition is combined with detergent ingredients to form a detergent composition and wherein said endoglucanase component is producible by a method comprising cultivating a host cell transformed with a recombinant DNA vector carrying a DNA sequence encoding said endoglucanase component or a precursor of said endoglucanase component, as well as DNA sequences encoding functions permitting the expression of the DNA sequence encoding the endoglucanase component, or a precursor thereof, in a culture medium under conditions permitting the expression of the endoglucanase component or precursor thereof and recovering the endoglucanase component from the culture.
 5. A dye transfer inhibiting composition according to claim 3 wherein said cellulase is producible by a species of Humicola.
 6. A detergent composition according to claim 4 wherein said host cell is a strain of a fungus selected from Tricloderuca or Aspergillus, or a yeast cell belonging to a strain of Hansenula or Saccharomyces.
 7. A detergent composition according to claim 4 wherein said host cell is a strain of a bacterium, e.g. Bacillus, Streptomyces or E. coli.
 8. A dye transfer inhibiting composition according to claim 1, wherein the hydrogen peroxide precursor is a perborate or percarbonate.
 9. A dye transfer inhibiting composition according to claim 8 wherein the level of said perborate is from 1 μM-10 mM of the wash solution.
 10. A dye transfer inhibiting composition according to claim 1, wherein said additional oxidizable substrate is selected from a metal ion, a halide ion or an organic compound such as a phenol, or a phenol sulfonate.
 11. A dye transfer inhibiting composition according to claim 1, wherein said enzymatic system capable of generating hydrogen peroxide is an oxidase selected from the group consisting of glucose oxidase, urate oxidase, galactose oxidase, alcohol oxidases, amine oxidases, amino acid oxidase and cholesterol oxidase.
 12. A dye transfer inhibiting composition according to claim 1, which is a detergent additive, in the form of a non-dusting granulate, a stabilized liquid, or a protected enzyme system.
 13. A dye transfer inhibiting composition according to claim 12 which is in granular form, compact granular form or liquid form.
 14. A process for inhibiting the transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed and/or rinsed together in a wash liquor, contacting said fabrics with a composition according to claim 1 above, the peroxidase being used at levels of from 0.01 to 100 mg/liter of wash solution, the level of hydrogen peroxide being from 0.001-5 mM of the wash solution, and the absolute level of additional oxidizable substrate being from 1 μM to 1 mM, and the cellulase being added at levels of from 0.005 to 40 mg enzyme protein/liter of wash solution. 